Viruses, Serums, Toxins, and Analogous Products; In Vitro Tests for Serial Release

DEPARTMENT OF AGRICULTURE

Animal and Plant Health Inspection Service

9 CFR Parts 101 and 113

[Docket No. 94-051-3]

RIN 0579-AA66

Viruses, Serums, Toxins, and Analogous Products; In Vitro Tests

for Serial Release

AGENCY: Animal and Plant Health Inspection Service, USDA.

ACTION: Final rule.

-----------------------------------------------------------------------

SUMMARY: We are amending the regulations to provide for the use of in

vitro potency tests when conducting immunoassays to determine the

relative antigen content (potency) of a serial of inactivated

veterinary biological product once immunogenicity is established using

host animal tests. Such tests would be conducted using unexpired

immunogenic reference preparations and parallel line assays, or other

methods which demonstrate linearity, specificity, and reproducibility

at least equivalent to the parallel line assay. Firms currently using

immunoassays which do not meet the standard in this amendment will have

2 years from the effective date of this final rule to update their

filed Outlines of Production. This amendment also changes the title of

the section and adds definitions of ``Master reference,'' ``Working

reference,'' ``Qualifying serial,'' and ``Immunogenicity'' to the

regulations.

The effect of this action is to standardize requirements for in

vitro immunoassay potency tests for inactivated products which cannot

be evaluated on the basis of virus titer or bacterial counts.

EFFECTIVE DATE: May 19, 1997.

FOR FURTHER INFORMATION CONTACT: Dr. David A. Espeseth, Director,

Center for Veterinary Biologics, Licensing and Policy Development, VS,

APHIS, 4700 River Road, Unit 148, Riverdale, MD 20737-1237, (301) 734-

8245.

SUPPLEMENTARY INFORMATION:

Background

The regulations pertaining to the testing of biologics provide that

no biological product shall be released (for sale) prior to the

completion of tests prescribed to establish the product to be pure,

safe, potent, and efficacious (9 CFR 113.5). Efficacy refers to the

specific ability of the product to effect the result for which it is

offered when used as recommended by the manufacturer. Tests to

establish efficacy include immunogenicity tests in host animals using

product which is manufactured according to specified requirements which

include specifications for antigen content and/or animal potency. If a

product has been tested for immunogenicity in animals and shown to

elicit the desired immune response, it should follow that subsequent

serials (batches) of the product manufactured to the same

specifications should also have the same effect. Based on this premise,

once immunogenicity is established in relation to a specific minimum

antigen content, it should no longer be necessary to test every

subsequent product serial for potency in animals if an evaluation of

the relative antigen content can be made by testing the serial or

subserial in an acceptable in vitro test system. Therefore, when

properly qualified and validated, in vitro immunoassays that determine

relative antigen content of a product can serve as acceptable

substitutes for potency tests that otherwise would need to be performed

in animals.

The regulations in 9 CFR 113.8 pertain to the use of in vitro tests

for determining the potency of serials and/or subserials of veterinary

biological products after required animal tests are completed. Prior to

this amendment, the in vitro test procedures prescribed in Sec. 113.8

were only applicable to products containing live microorganisms. With

these amendments Sec. 113.8 will be applicable to both live and

inactivated products.

On May 17, 1995, we published in the Federal Register (60 FR 26381-

26384, Docket No. 94-051-1) a proposal to amend the regulations

regarding the use of in vitro potency tests in place of animal tests

for immunogenicity. The proposed rule provided for the use of a

parallel line assay, or other valid method, and an unexpired reference

preparation in an in vitro immunoassay for relative antigen content to

determine the potency of a serial of inactivated product. In proposing

the parallel line assay or equivalent valid method and the use of an

unexpired reference as a standard for in vitro immunoassay potency

tests for serial release, APHIS did not intend to preclude the

validation of existing in vitro immunoassays or the adoption of

technological advances in antigen quantitation.

We solicited comments concerning our proposal for 90 days ending

August 15, 1995. We extended the comment period an additional 30 days

ending September 14, 1995 (60 FR 36743-36744, Docket No. 94-051-2, July

18,

[[Page 19034]]

1995). We also announced that we would be having a public hearing on

August 1, 1995, in Ames, IA, to have further discussion related to in

vitro testing by interested persons. We received comments from four

licensed manufacturers, and a national trade association representing

U.S. manufacturers of animal health products. Three comments from

biologics producers were received at the public hearing on August 1,

1995, in Ames, IA. While generally supportive of in vitro immunoassay

tests for determining the relative antigen content and thereby the

potency of products, most commenters suggested changes in one or more

sections as proposed. Others suggested that the comment period be

extended and the proposal be submitted to negotiated rulemaking. We

carefully considered all of the comments we received. They are

discussed below.

Analysis of Comments and APHIS' Response

Four commenters requested that the comment period be further

extended for 10 months beyond September 14, 1995, and that negotiated

rulemaking be initiated. In response to this comment, APHIS notes that

the history of this rulemaking began with a proposed rule published on

May 17, 1995 (60 FR 26381-26384, Docket No. 94-051-1). The comment

period of 90 days was extended to 120 days until September 14, 1995, in

response to a request for an extension from a national trade

association (See 60 FR 36743, July 18, 1995, Docket No. 94-051-2). In

addition, a public hearing was held on In Vitro Potency Testing on

August 1, 1995, in Ames, IA to obtain further comment on this topic.

Contrary to the commenters' request, the comment period cannot be

further extended for negotiated rulemaking because the initiation of

negotiated rulemaking necessitates the withdrawal of the current

proposal and the proposal of another rule after the conclusion of the

negotiated rulemaking. APHIS believes that the publication of a final

rule with appropriate consideration of responses and comments would be

a more efficient way of handling this matter and would allay concerns

and clarify issues raised by the commenters. Therefore, the request for

further extension of the comment period and initiation of negotiated

rulemaking is not granted.

Two commenters expressed concern that by specifying that in vitro

immunoassays used to determine relative antigen content be parallel

line assays, APHIS would be imposing a requirement which would not

allow the industry to take advantage of technological advances that are

occurring in the area of antigen quantitation. APHIS proposed the

parallel line assay as a standard for immunoassay tests for relative

antigen content. Assay formats which are equivalent to or exceed the

parallel line assay standard could have been used as provided for in 9

CFR 113.4. In response to these comments, however, APHIS has amended

Secs. 101.5(q) and 113.8(a) in the final rule to provide specifically

for the use of other valid methods for determining relative antigen

content which demonstrate linearity, specificity, and reproducibility

at least equivalent to the parallel line assay.

Five commenters recommended amending the rule to allow laboratory

animal tests and antibody titers that have been correlated to host

animal protection to be used to requalify or extend the dating of

reference preparations. One of the commenters pointed out that the

proposed standard requirement for Escherichia coli (E. coli) bacterins

(59 FR 51390-51392, October 11, 1994) which also uses a parallel line

immunoassay to test for potency, includes such a provision. In

addition, the commenter interpreted the E. coli standard requirement to

imply that in vitro assays may be used in place of reference

requalification in host animals. In response to the commenter, APHIS

agrees that the proposed E. coli standard requirement allows antibody

titers and laboratory animal studies, previously correlated to

protection, to be used to requalify reference preparations. These same

provisions were available under the proposal to amend Secs. 101.5 and

113.8 (See proposed terminology in Sec. 101.5(o) which provides for

direct or indirect correlation of potency to host animal

immunogenicity). However, by specifying in proposed Sec. 101.5(q)(1)

that Qualifying Serials used to requalify or extend the dating of a

Master Reference shall be ``tested for immunogenicity in host

animals,'' APHIS may have inadvertently implied that laboratory animal

tests could not be used for reference requalification. This was not the

intent of the proposed regulation. In response to the commenter, the

final rule has been amended in Sec. 101.5(q)(1) to clarify the

definition of Qualifying Serial to provide for the use of procedures

acceptable to APHIS which will include antibody titers and laboratory

animal testing along with host animal immunogenicity for reference

requalification.

In response to the comment regarding the use of in vitro assays to

requalify or extend the dating of a reference in place of performing

studies in animals, in vitro tests may not be substituted for animal

tests for reference requalification. The proposed E. coli standard

requirement stated that an in vitro procedure may be used to monitor

the potency of the Master Reference for indication of decline, but

specified that the reference must be requalified when a decline in

potency is detected. As proposed in the proposed E. coli standard

requirement, the immunogenicity of Qualifying Serials used in reference

requalification studies may be based on host animal studies (challenge

or antibody titer) or laboratory animal studies as provided in

protocols acceptable to APHIS. Therefore, to clarify these points and

to eliminate the apparent inconsistency between the two proposed rules,

APHIS is amending Sec. 113.8(d)(2) pertaining to in vitro testing to

include a monitoring provision and to clarify that: (1) The monitoring

procedure can only be used to monitor the unexpired reference to detect

when a decline in potency has occurred between requalification

intervals, and (2) to specify that, if such monitoring procedures

indicate the potency of the reference is declining, the reference must

be requalified either by testing a Qualifying Serial in host animals or

by providing other evidence of reference immunogenicity, e.g., antibody

titers or laboratory animal test data previously correlated to host

animal protection, or a new reference must be prepared and qualified.

In vitro monitoring, however, would not be a substitute for reference

requalification at the end of product dating.

One commenter suggested amending Sec. 113.8(c)(5) to include a

provision to allow a firm to declare a potency test with valid lines a

``no test'' if the firm does not have confidence in the test result.

APHIS does not agree that it would be appropriate to declare such a

test a ``no test''. The regulation, as proposed, allows a firm to

retest a serial two times when the initial test shows that potency is

less than the required minimum potency. The commenter's suggestion,

however, would make potency testing subjective and allow a firm to

disregard valid results that are not consistent with a desired outcome.

Conceivably, a serial with unsatisfactory test results could be

retested indefinitely. In response to this comment, APHIS has clarified

provisions for the retesting of such serials and permitted up to three

retests to be performed. Provisions have also been added to permit the

potency test to be repeated under certain specified conditions.

Two commenters requested that firms be allowed more than two years

to

[[Page 19035]]

convert currently approved in vitro immunoassays that are described in

filed Outlines of Production that are not parallel line assays, to

parallel line assays or to another method which demonstrates linearity,

specificity, and reproducibility at least equivalent to the parallel

line assay. They believed that the two-year timetable will have a

negative impact on new product development, and therefore result in

fewer new products on the market. In response, APHIS realizes that some

firms may require more than two years to convert to parallel line

assays or other valid methods. However, two years from the effective

date of the final rule should be adequate time for most firms to

validate their immunoassays and requalify references for existing

products, considering that a single reference requalification procedure

may be applicable to several different products. Also, those firms

experiencing difficulty in meeting the time period may be granted

additional time, if justified, by requesting an extension as provided

in the regulations. Therefore, no change to the regulations is made in

response to these comments.

One commenter requested that the definition of ``Master Reference''

in Sec. 101.5(o) be amended to include options and directions for

stabilizing and storing reference preparations. The commenter believed

that this will result in more options for treating the references.

APHIS does not agree that the rule needs to be amended. The definition

of a ``Master Reference'' does not limit the options available to firms

when it comes to stabilizing, storing, lyophilizing, or freezing Master

References provided that such procedures are described in the filed

Outline of Production. Specifying such procedures in the definition,

however, would limit the industry to the procedures defined. Since the

proposed definition does not limit the available options, no change to

the regulations is made in response to this comment.

Another commenter requested clarification of proposed Sec. 101.5(p)

of the regulations. The commenter inquired if a purified antigen

preparation could serve as the Working Reference. As proposed in

Sec. 101.5(p) of the regulations, the Working Reference may be the

Master Reference, and since the Master Reference may be a purified

preparation of the protective immunogen (antigen), it follows that a

purified antigen can serve as the Working Reference. Therefore, no

change to the regulations is made in response to this comment.

One commenter recommended amending proposed Sec. 101.5(q)(1) of the

rule to require Qualifying Serials for reference requalification to be

produced at the minimum antigen level specified in the Outline of

Production instead of specifying that the geometric mean relative

potency not exceed 1.0 when compared to the Master Reference. The

commenter reasoned that, by specifying that the amount of antigen in

the Qualifying Serial not exceed the amount of antigen contained in the

Master Reference, the antigen level contained in the Master Reference

is a more appropriate benchmark (measure of protection) than is the

antigen content specified in the Outline of Production. The commenter

believed that the amount of antigen specified in the Outline of

Production should establish the antigen requirement for the Qualifying

Serial. APHIS does not agree with the commenter's recommendation. In

measuring relative potency, the antigen level used to demonstrate host

animal protection becomes the benchmark by which other serials are

measured and is the level of antigen to be contained in a Qualifying

Serial that is used to determine if the Master Reference is still

protective and therefore eligible for continued use in the potency

assay. The commenter's recommendation of using a regular production

serial and devising a calculation procedure to show antigen equivalency

is an indirect method that was considered by APHIS and determined to be

inappropriate and less meaningful than the provision in the APHIS

proposal. Therefore, no change to the regulations is made in response

to this comment.

Two commenters expressed confusion regarding proposed

Secs. 113.8(a)(4) (i) and (ii) of the rule. The commenters noted that

although Sec. 113.8(a)(4) refers to in vitro methods for determining

the potency of inactivated products, the cited examples, i.e.,

determining log10 virus titer and determining the live bacterial

count only apply to live products. APHIS agrees that the wording of

proposed Sec. 113.8(a)(4) is contradictory and has amended the final

rule, eliminating the contradictory sections, by incorporating the

provisions of Sec. 113.8(a)(4) into Sec. 113.8(a)(3) as follows:

(3) Establishing a satisfactory potency test for the product in

accordance with the following provisions:

(i) Potency of live products may be determined by log10

virus titer or determining the live bacterial count based on the

protective dose used in the Master Seed immunogenicity test plus an

adequate overage for adverse conditions and test error; and

(ii) Potency for inactivated products may be determined using

tests for relative antigen content by comparing the antigen content

of the test serial to a reference preparation using a parallel line

immunoassay or equivalent method which measures linearity,

specificity, and reproducibility in a manner acceptable to APHIS.

One commenter requested that the phrase ``an appropriate

difference'' referred to in proposed Sec. 113.8(b)(5) be further

defined. Proposed Sec. 113.8(b)(5) pertains to in vitro potency tests

for live vaccines in which potency is measured in terms other than

log10 virus titer or live bacterial counts, e.g., Marek's Disease

vaccines in which potency is measured in terms of plaque forming units

(PFU). Generally, an appropriate difference pertains to how a serial is

determined to have satisfactory potency when the initial potency test

determines that the serial contains less than the number of PFU's

specified in the Outline of Production (OP) or standard requirement and

the manufacturer elects to retest the serial to rule out test system

error as the cause of the unsatisfactory test result. In accordance

with Sec. 113.8(b)(5), the manufacturer must specify in the OP the

difference between the average PFU count obtained in the retest and the

PFU count obtained in the initial test so that the initial test may be

considered a result of test system error. The commenter did not suggest

what this appropriate difference in PFU or organism count should be.

APHIS has noted that the appropriate difference between test results

may be different for each product and this is the reason the proposed

rule specified that this value should be placed in the product Standard

Requirement or filed OP. From data submitted to APHIS, however, it is

also noted that an acceptable guideline for determining such

appropriate difference would be if the difference between the average

PFU count obtained in the retest and the count obtained in the initial

test exceeds 20 per cent. However, because no specific value was

proposed by the commenter, and there is a need to address specific

product differences, no change to the regulation is made in response to

this comment.

One commenter proposed that tests for relative antigen content

which cannot be termed satisfactory or unsatisfactory should be called

``no tests'' and be eligible for unlimited retesting without prejudice.

In response, APHIS points out that Sec. 113.8(c)(1) of the regulation

classifies a test that results in no valid lines as a ``no test''.

Typically, this designation is used when a deficiency in the test

system renders an invalid test result which is

[[Page 19036]]

unsuitable for reaching a conclusion regarding the potency of a serial;

such serials may be retested. An equivocal test as that test is used in

Sec. 113.8(c)(2), is a test that results in valid lines which are not

parallel. Therefore, the test is considered inconclusive and the serial

cannot be termed satisfactory or unsatisfactory. In order to clarify

the proper handling and disposition of serials of product with

equivocal test results, APHIS has amended Secs. 113.8 (c) (4) and (5)

regarding the retest of serials with equivocal test results due to a

lack of parallelism by specifying (1) the number of times such serials

may be retested and, (2) the disposition of the serial based on the

results of the retest.

Four comments were received related to proposed Sec. 113.8(d)(2).

The commenters requested that: (1) Stabilized Master References be

allowed to serve as Working References; (2) Master References be

allowed an initial dating period at least twice as long as that allowed

for a regular serial of product; and (3) Frozen references be allowed

an initial expiration dating of 5 years, provided that they are

monitored by in vitro methods. In response to these comments regarding

item (1), APHIS notes that proposed Sec. 101.5(o) of the regulation

specifies that the Master Reference may be used as the Working

Reference. Regarding item (2), proposed Sec. 113.8(d)(2) specifies that

the dating of the reference shall be equal to the dating of the product

or as supported by data acceptable to APHIS. Stability can be

demonstrated by repeat testing of the reference over time or by

demonstrating that the reference has maintained immunogenicity after

being stored for a period of time equal to or greater than the dating

period requested. Regarding item (3), allowing longer dating for

references based on special treatments or storage conditions may be

justified if such treatments or storage conditions are better able to

maintain the stability of the reference. Section 113.8(d)(2) provides

for determining the stability of the reference on the basis of

confirming the immunogenicity in a manner acceptable to APHIS. This

would include data from a stabilized monitored reference demonstrating

stability in a manner acceptable to APHIS. Therefore, a reference may

be allowed to have an initial dating longer than that for a regular

production serial, provided that the request for the longer initial

dating is supported by appropriate preliminary data and provides for

monitoring stability to determine when the potency of the reference

starts to decline and for taking appropriate steps to requalify or

replace such a reference.

In response to the commenters, APHIS has amended the regulations to

allow frozen references an initial dating period of 5 years, provided

that the request for such initial dating is supported by preliminary

data and a frozen storage protocol, including monitoring procedures,

acceptable to APHIS. As amended, Sec. 113.8(d)(2) reads as follows:

(d)(2) * * * The lot of reference used to determine antigenic

content shall have an initial dating period equal to the dating of

the product or as supported by data acceptable to APHIS, except that

frozen references may have an initial dating of up to 5 years,

Provided, That the request for dating of frozen references beyond

the dating of the product is supported by preliminary data

acceptable to APHIS and includes provisions for monitoring the

stability of the reference to determine when the potency starts to

decline and for taking the appropriate steps to requalify a

reference with declining potency either by testing a Qualifying

Serial in host animals or by providing other evidence of

immunogenicity, e.g., antibody titers or laboratory animal test data

previously correlated to host animal protection in a manner

acceptable to APHIS. Prior to the expiration date, such reference

may be granted an extension of dating, Provided, That its

immunogenicity has been confirmed using a Qualifying Serial of

product in a manner acceptable to APHIS. * * *

APHIS received two comments on proposed Sec. 101.5(q)(2) inquiring

into the rationale for requiring the qualifying serial used to extend

the dating of a Master Reference to be prepared within 6 months of

initiating a requalification test. The commenters believed that the 6

month restriction limited their options relating to production

schedules and antigen manufacture. APHIS proposed the 6 month

restriction as a means of assuring that qualifying serials used to

extend the dating of a reference would be representative of the firm's

current production method. APHIS agrees with the commenters regarding

the potential restrictive aspects of the 6 month requirement and has

amended Sec. 101.5(q)(2) in response to the comment to be more

consistent with our intent as follows:

(2) Qualifying serials used to requalify or extend the dating

period of a Master Reference shall be determined to be immunogenic

in accordance with methods deemed appropriate by APHIS as provided

in paragraph (a)(1) of this section, and, in addition, shall be

within their permitted dating period and have been prepared in

accordance with the production method described in the currently

filed Outline of Production.

APHIS received one comment requesting clarification of proposed

Sec. 113.8(d)(1) concerning confirmation of the protective dose

established for live products in the Master Seed immunogenicity test

after three years. In response to this comment, confirming the accuracy

of the protective dose for live products three years after completion

of a satisfactory immunogenicity test is specified in the Standard

Requirements for live viral vaccines, and in the filed Outline of

Production for products where standards have not been codified.

Including a reference to this requirement for live viral vaccines in

Sec. 113.8(d)(1) corrects an omission and provides notification of the

requirement to those unfamiliar with this provision of the regulations.

As specified in the codified requirements for individual live viral

vaccines, only one retest is required. No change to the regulations is

made in response to this comment.

We received two comments regarding the definition of a ``Qualifying

Serial'' in Sec. 101.5(q)(1). The commenter expressed concern that

limiting a qualifying serial to a relative potency, when compared to

the Master Reference, of not greater than 1.0 is too restrictive. The

commenters suggested that the normal tolerance limits of 15

per cent for parallel line immunoassays could cause a Qualifying Serial

set at 1.0 to be as low as 0.85, which means that it may not pass a

requalification test in animals. APHIS does not agree that requiring

the Qualifying Serial to have a mean relative potency of not greater

than 1.0 is too restrictive. As the commenter is probably aware, test

assay variation is to be expected. Usually, a manufacturer will

optimize the test system to determine how much variation is normal, and

adjust the antigen levels so that the risk of failing a requalification

test in animals is minimized. The alternative would require APHIS to

include tolerance limits in the regulations. APHIS does not agree that

such tolerance limits are necessary. The individual manufacturers can

optimize antigen levels based on their individual experiences with test

assay variation to assure that a Qualifying Serial with a mean relative

potency of not greater than 1.0 will pass the requalification test in

animals. No change to the regulations is made in response to this

comment.

Therefore based on the rationale set forth in the proposed rule and

in this document, we are adopting the provisions of the proposal as a

final rule, with the changes discussed in this document.

[[Page 19037]]

Executive Order 12866 and Regulatory Flexibility Act

This rule has been reviewed under Executive Order 12866. The rule

has been determined to be not significant for purposes of Executive

Order 12866, and therefore, has not been reviewed by the Office of

Management and Budget.

This amendment allows any valid in vitro immunoassay to be used in

determining the relative antigen content of an inactivated veterinary

biological product, provided that it satisfies the parallel line

criteria or demonstrates linearity, specificity, and reproducibility

equivalent to the parallel line assay using an unexpired reference

preparation. This amendment affects all licensed manufacturers of

veterinary biologicals utilizing in vitro relative potency immunoassays

for determining the potency of animal biological products. There are

currently approximately 118 veterinary biologics establishments that

may be affected by this rule. According to the Small Business

Administration regulations, most of them would be classified as small

entities. The majority of these establishments currently utilize in

vitro relative potency tests to release serials of veterinary

biological products. Since potency testing is already required under

Sec. 113.5 of the regulations and since this rule does not require the

use of in vitro relative potency tests, any additional cost imposed by

the validity requirements specified in this rule should be minimal. In

the absence of a standard requirement prescribing a specific potency

test for inactivated products, the firms develop a potency test

suitable for their product, and designate such tests in the outline of

production that is filed with APHIS. Currently, firms are using host

animal tests, laboratory animal tests, and a variety of in vitro

immunoassays as potency tests for products. This rule does not restrict

the firm's discretion to choose the most appropriate test for its

product. The rule only prescribes validity requirements for in vitro

immunoassays for relative potency. The overall effect of this amendment

will be to standardize in vitro immunoassays that are used to determine

the potency of inactivated veterinary biological products.

Under these circumstances, the Administrator of the Animal and

Plant Health Inspection Service has determined that this action will

not have a significant economic impact on a substantial number of small

entities.

Executive Order 12372

This program/activity is listed in the Catalog of Federal Domestic

Assistance under No. 10.025 and is subject to Executive Order 12372,

which requires intergovernmental consultation with State and local

officials. (See 7 CFR part 3015, subpart V.)

Executive Order 12988

This final rule has been reviewed under Executive Order 12988,

Civil Justice Reform. It is not intended to have retroactive effect.

This rule would not preempt any State or local laws, regulations, or

policies, unless they present an irreconcilable conflict with this

rule. There are no administrative procedures which must be exhausted

prior to a judicial challenge to the provisions of this rule.

Paperwork Reduction Act

In accordance with the Paperwork Reduction Act of 1995 (44 U.S.C.

3501 et seq.), the information collection or recordkeeping requirements

included in this rule have been approved by the Office of Management

and Budget (OMB), and there are no new requirements. The assigned OMB

control number is 0579-0013.

Regulatory Reform

This action is part of the President's Regulatory Reform

Initiative, which, among other things, directs agencies to remove

obsolete and unnecessary regulations and to find less burdensome ways

to achieve regulatory goals.

List of Subjects

9 CFR Part 101

Animal biologics.

9 CFR Part 113

Animal biologics, Exports, Imports, Reporting and recordkeeping

requirements.

Accordingly, 9 CFR parts 101 and 113 are amended as follows:

PART 101--DEFINITIONS

1. The authority citation for part 101 continues to read as

follows:

Authority: 21 U.S.C. 151-159; 7 CFR 2.22, 2.80, and 371.2(d).

2. Section 101.5 is amended by adding new paragraphs (o), (p), (q),

and (r) to read as follows:

Sec. 101.5 Testing terminology.

* * * * *

(o) Master reference. A Master Reference is a reference whose

potency is correlated, directly or indirectly, to host animal

immunogenicity. The Master Reference may be used as the working

reference in in vitro tests for relative potency. The Master Reference

may also be used to establish the relative potency of a serial of

product used in requalification studies and to establish the relative

potency of working references. The preparation of a Master Reference as

described in a filed Outline of Production may be:

(1) A completed serial of vaccine or bacterin prepared in

accordance with a filed Outline of Production;

(2) A purified preparation of a protective immunogen or antigen; or

(3) A nonadjuvanted harvested culture of microorganisms.

(p) Working reference. A Working Reference is the reference

preparation that is used in the in vitro test for the release of

serials of product. Working References may be:

(1) Master References; or

(2) Serials of product that have been prepared and qualified, in a

manner acceptable to Animal and Plant Health Inspection Service for use

as reference preparations.

(q) Qualifying serial. (1) A serial of biological product used to

test for immunogenicity when the Master or Working Reference is a

purified antigen or nonadjuvanted harvest material. Qualifying serials

shall be produced in accordance with the filed Outline of Production,

tested for immunogenicity in accordance with methods deemed appropriate

by the Animal and Plant Health Inspection Service, and have a geometric

mean relative potency, when compared to the Master Reference, of not

greater than 1.0 as established by: independent parallel line assays

with five or more replicates; or other valid assay methods for

determining relative antigen content which demonstrate linearity,

specificity, and reproducibility at least equivalent to the parallel

line assay and are acceptable to the Animal and Plant Health Inspection

Service.

(2) Qualifying serials used to requalify or extend the dating

period of a Master Reference shall be determined to be immunogenic in

accordance with methods deemed appropriate by the Animal and Plant

Health Inspection Service as provided in paragraph (a)(1) of this

section, and, in addition, shall be within their permitted dating

period and have been prepared in accordance with the production method

described in the currently filed Outline of Production.

(r) Immunogenicity. The ability of a biological product to elicit

an immune response in animals as determined by test methods or

procedures acceptable

[[Page 19038]]

to the Animal and Plant Health Inspection Service.

PART 113--STANDARD REQUIREMENTS

3. The authority citation for part 113 continues to read as

follows:

Authority: 21 U.S.C. 151-159; 7 CFR 2.22, 2.80, and 371.2(d).

4. Section 113.8 is amended as follows:

a. The section heading is revised to read as set forth below.

b. Paragraph (a) is revised to read as set forth below.

c. Paragraph (b) introductory text is revised to read as set forth

below.

d. Paragraph (b)(5) is revised to read as set forth below.

e. Paragraph (c) is redesignated as paragraph (e) and new

paragraphs (c) and (d) are added to read as set forth below.

f. In redesignated paragraph (e), in the introductory text, the

reference to ``paragraph (b)'' is removed and ``paragraphs (b) and

(c)'' are added in its place. In redesignated paragraph (e)(4), the

reference to ``paragraphs (c)(1),'' is removed and ``paragraphs

(e)(1),'' is added in its place.

Sec. 113.8 In vitro tests for serial release.

(a) Master Seed which has been established as pure, safe, and

immunogenic shall be used for preparing seed for production as

specified in the Standard Requirements or in the filed Outline of

Production. The Administrator may exempt a product from a required

animal potency test for release when an evaluation can, with reasonable

certainty, be made by:

(1) Subjecting the master seed to the applicable requirements

prescribed in Secs. 113.64, 113.100, 113.200, and 113.300;

(2) Testing the Master Seed for immunogenicity in a manner

acceptable to the Animal and Plant Health Inspection Service (APHIS);

(3) Establishing satisfactory potency for the product in accordance

with the following provisions:

(i) Potency for live products may be determined by log10 virus

titer or determining the live bacterial count based on the protective

dose used in the Master Seed immunogenicity test plus an adequate

overage for adverse conditions and test error; and

(ii) Potency for inactivated products may be determined using tests

for relative antigen content by comparing the antigen content of the

test serial to a reference preparation using a parallel line

immunoassay or equivalent method which measures linearity, specificity,

and reproducibility in a manner acceptable to APHIS.

(b) In the case of live products, each serial and subserial of

desiccated product derived from an approved Master Seed and bulk or

final container samples of each serial of completed liquid product

derived from an approved Master Seed shall be evaluated by a test

procedure acceptable to APHIS. On the basis of the results of the test,

as compared with the required minimum potency, each serial and

subserial shall either be released to the firm for marketing or

withheld from the market. The evaluation of such products shall be made

in accordance with the following criteria:

* * * * *

(5) Exceptions. When a product is evaluated in terms other than

log10 virus titer or organism count, an appropriate difference

between the average potency value obtained in the retests and the

potency value obtained in the initial test shall be established for use

in paragraphs (b)(3) and (b)(4) of this section to evaluate such

products and shall be specified in the product Standard Requirement or

filed Outline of Production.

(c) In the case of inactivated products, bulk or final container

samples of completed product from each serial derived from an approved

Master Seed, shall be evaluated for relative antigen content (potency)

as compared with an unexpired reference by a parallel line immunoassay

or other procedure acceptable to APHIS.\1\ Firms currently using

immunoassays which do not satisfy this requirement shall have 2 years

from the effective date of the final rule to update their filed

Outlines of Production to be in compliance with this requirement unless

granted an extension by the Administrator based on a showing by the

firm seeking the extension that they have made a good faith effort with

due diligence to achieve compliance. On the basis of the results of

such test procedures, each serial that meets the required minimum

potency shall be released to the firm for marketing; each serial not

meeting the required minimum potency shall be withheld from the market.

The evaluation of such products shall be made in accordance with the

following criteria:

---------------------------------------------------------------------------

\1\ A method for evaluating relative antigen content,

Supplemental Assay Method 318, and relative potency calculation

software are available from the United States Department of

Agriculture, Animal and Plant Health Inspection Service, Veterinary

Services, National Veterinary Services Laboratories, Center for

Veterinary Biologics--Laboratory, 1800 Dayton Road, P. O. Box 844,

Ames, Iowa 50010.

---------------------------------------------------------------------------

(1) A test that results in no valid lines is considered a ``no

test'' and may be repeated.

(2) An initial test (test 1) that results in valid lines that are

not parallel is considered a valid equivocal test. Release of the

serial may not be based on such test since the result cannot be termed

``satisfactory'' or ``unsatisfactory.''

(3) If the initial test (test 1) shows that potency equals or

exceeds the required minimum potency, the serial is satisfactory

without additional testing.

(4) If the initial test (test 1) is an equivocal test due to lack

of parallelism, the serial may be retested up to three times (tests 2,

3, and 4) with disposition to be as specified in paragraphs (c)(4)(i)

and (ii) of this section; Provided, That, if the serial is not retested

or the other provisions of this section are not satisfied, the serial

shall be deemed unsatisfactory.

(i) If: The first retest (test 2) following an initial equivocal

test; the second retest (test 3) following two consecutive equivocal

tests (tests 1 and 2); or the third retest (test 4) following three

consecutive equivocal tests (tests 1, 2, and 3) shows that the potency

equals or exceeds the required minimum potency, the serial is

satisfactory.

(ii) If the first retest (test 2) following an initial equivocal

test shows that potency is less than the required minimum potency,

disposition of the serial will be based on the outcome of retests 2 and

3 (tests 3 and 4) as follows: if either retest (test 3 or 4) shows that

potency is less than the required minimum potency, the serial is

unsatisfactory. If either retest 2 or retest 3 (tests 3 or 4) is an

equivocal test, or in the event that each retest (tests 2, 3, and 4)

following an initial equivocal test is also an equivocal test, the

accumulated test results shall be considered indicative of a lack of

potency and release of the serial withheld. In which case, the licensee

may submit data confirming the continued validity of the test system to

APHIS for review and approval. If the data are acceptable to APHIS, the

potency test may be repeated by the firm, subject to the provisions

specified in paragraphs (i) and (ii) and confirmatory testing by APHIS.

(5) If the initial test (test 1) shows that potency is less than

the required minimum potency, the serial may be retested a minimum of

two times (tests 2 and 3) but not more than three times (tests 2, 3,

and 4) with disposition as specified in paragraphs (c)(5) (i) and (ii)

of this section; Provided, That, if the

[[Page 19039]]

serial is not retested or the other provisions of this section are not

satisfied, the serial shall be deemed unsatisfactory.

(i) If two consecutive retests (tests 2 and 3) show that potency of

the serial equals or exceeds the required minimum potency, the serial

is satisfactory. If one of the two retests (test 2 or 3) shows that the

potency is less than the required minimum potency, the serial is

unsatisfactory.

(ii) If one of the retests (tests 2 or 3) shows that the potency

equals or exceeds the required minimum potency and the other retest

(test 2 or 3) is an equivocal test, a third retest (test 4) may be

performed. If the third retest (test 4) shows that the potency of the

serial equals or exceeds the required minimum potency, the serial is

deemed satisfactory. If both retests (tests 2 and 3) or if the third

retest (test 4) is an equivocal test, the accumulated test results

shall be considered indicative of a lack of potency and release of the

serial withheld, in which case the licensee may submit data confirming

the continued validity of the test system to APHIS for review and

approval. If the data are acceptable to APHIS, the potency test may be

repeated by the firm, subject to the provisions specified in paragraphs

(c)(4) (i) and (ii) and (c)(5) (i) and (ii) of this section, and

confirmatory testing by APHIS.

(d) Repeat immunogenicity tests. (1) The accuracy of the protective

dose established for live products in the Master Seed immunogenicity

test and defined as live virus titer or live bacterial count shall be

confirmed in 3 years in a manner acceptable to APHIS, unless use of the

lot of Master Seed previously tested is discontinued.

(2) All determinations of relative antigen content using parallel

line immunoassays or equivalent methods shall be conducted with an

unexpired reference. The lot of reference used to determine antigenic

content shall have an initial dating period equal to the dating of the

product or as supported by data acceptable to APHIS, except that frozen

references may have an initial dating of up to 5 years, Provided, That

the request for dating of the frozen references beyond the dating of

the product is supported by preliminary data acceptable to APHIS and

includes provisions for monitoring the stability of the reference to

determine when the potency starts to decline and for taking the

appropriate steps to requalify a reference with declining potency

either by testing a Qualifying Serial in host animals or by providing

other evidence of immunogenicity, e.g., antibody titers or laboratory

animal test data previously correlated to host animal protection in a

manner acceptable to APHIS. Prior to the expiration date, such

reference may be granted an extension of dating, Provided, That its

immunogenicity has been confirmed using a Qualifying Serial of product

in a manner acceptable to APHIS. The dating period of the Master

Reference and Working Reference may be extended by data acceptable to

APHIS if the minimum potency of the Master Reference is determined to

be adequately above the minimum level needed to provide protection in

the host animal. If a new Master Reference is established, it shall be

allowed an initial dating period equal to the dating of the product or

as supported by data acceptable to APHIS, except that frozen references

may have an initial dating period of 5 years, or as supported by data

acceptable to APHIS. Prior to the expiration date, such reference may

be granted an extension of dating by confirming its immunogenicity

using a Qualifying Serial of product.

* * * * *

Done in Washington, DC, this 15th day of April 1997.

Donald W. Luchsinger,

Acting Administrator, Animal and Plant Health Inspection Service.

[FR Doc. 97-10100 Filed 4-17-97; 8:45 am]

BILLING CODE 3410-34-P